Excessive epidermal growth factor receptor (EGF-R) signaling is linked to multiple cancers. Understanding the mechanisms of action of endogenous EGF-R suppressors may lead to novel antitumor therapies. The E3/ubiquitin ligase Cbl is an endogenous EGF-R signaling suppressor that enhances EGF-R ubiquitination, downregulation and lysosomal degradation. Cbl also mediates the degradation of the endocytic trafficking regulators Sprouty2 and Hrs. All of these functions depend upon the integrity of a Cbl domain known as the RING finger (RF) tail. We have noted sequence similarity between the Cbl and Mdm2 RF tails. The Cbl- related RF tail residues of Mdm2 enhance Mdm2's oligomerization and E3 activity. Here, we hypothesize that Cbl's RF tail regulates Sprouty2, Hrs, and EGF-R fate by enhancing their ubiquitination via Cbl oligomerization. Furthermore, we postulate that RF tail-dependent phosphorylation of Hrs is necessary for efficient Hrs and EGF-R degradation. Finally, we propose that RF tail-dependent Hrs phosphorylation alters endosome fusion activity or the composition of endosomal sorting machinery/EGF-R complexes, thereby altering the degradative trafficking of EGF-R. Aim I: wild type and RF tail mutant Cbl proteins will be compared in vitro for their ability to: a) form and maintain Cbl oligomers;b) interact with the E2 protein UbcH7;and c) enhance target protein ubiquitination. Structure-function studies in a cellular context will test whether Cbl residues that may interact with the RF tail at oligomer interfaces are functionally important for Sprouty2 and EGF-R ubiquitination/ degradation. Finally, wt Cbl and RF tail/other Cbl mutants will be compared for their impact on receptor trafficking and signaling. Aim II: Cbl mutants and an shRNA Hrs knockdown/reconstitution approach will be used to test the hypothesis that Hrs tyrosine phosphorylation controls EGF-R signaling, trafficking, and degradation. Aim III: wt and mutant Hrs proteins will be used to test specific mechanisms through which Hrs tyrosine phosphorylation sites may regulate EGF-R fate, including the modulation of endosome fusion and protein-protein interactions. Overexpression of EGF receptors is associated with a variety of tumors of epithelial origin, including those of the breast, pancreas, colon, bladder, prostate, head and neck, and lung. The study of endogenous suppressors of EGF-R signaling could reveal novel molecular targets for therapeutic intervention in these cancers. For this reason, defining how Cbl and Hrs interact to regulate EGF-R signaling and degradation is an important goal in cancer research.